i want to experimentally analyze the Tm of a short oligonucleotide probe (26 bp). first of what annealing buffer should i use? (i have the exact complementary sequence too.) and also i want to use the rotor q gene device. i program it to have an hybridization and melting curve analysis step. but i get various peaks. ( i used a syber green master mix, should i only use syber green or any other dsDNA dye alone? does the taq polymerase in the mastermix interfere with my melt curve analysis result?) reading OD at different temperature intervals is not sensitive and not applicable in our lab since the nanodrop device isn't really sensitive. and uses alot of the probe. should i also do a PAGE electrophoresis analysis of the melt curve analysis product? how about i only program rotor q gene device to melt the annealed products at the specified Tm ( if the multiple peak problem resolves) and run the PAGE electrophoresis. to see how much ssDNA and dsDNA i have. and also to check the specificity i have two other oligonucleotides with 2 and 3 mismatches to be synthesized as well.
eagerly looking forward to suggestions. thanks _______________________________________________ Methods mailing list [email protected] http://www.bio.net/biomail/listinfo/methods
